Extract of Rehmannia glutinasa Libosch. for reducing blood glucose and lipid levels and treating hematologic diseases, and methods for preparing the same

ABSTRACT

The present invention relates to a  Rehmannia glutinasa Libosch . extract for reducing blood glucose and lipid levels and treating hematologic diseases such as leukemia, and methods for preparing the same. The extract is prepared by subjecting fresh  Rehmannia glutinasa Libosch . to flux extraction, concentrating the resultant extract, dissolving the concentrated extract with water, loading the obtained solution on a column packed with macroporous adsorption resins, eluting the column with ethanol, concentrating and drying the eluate. The extract can be made into clinically acceptable forms of dosage by pharmaceutically conventional measures, including capsule, tablet, pill, granule, dropping pill and so on. The extract is stable and has catalpol content up to 90-98%. The preparing method is simple to operate and applicable to industrial-scale production.

TECHNICAL FIELD

The present invention relates to an extract from Chinese materia medica(CMM) and methods for preparing the same. More particularly, the presentinvention relates to an effective fraction of Rehmannia glutinasaLibosch. for treating hyperglycemia, hyperlipidemia and hematologicdiseases like leukaemia, its preparation and methods for preparing thesame.

BACKGROUND ART

With the development of social economy, great changes have taken placein people's life style and dietary choices, which thus results indramatic changes in spectrum of diseases, and chronic non-infectiousdiseases are becoming prominent problems endangering people's physicaland spiritual health. Among these diseases, hyperglycemia,hyperlipidemia and leukaemia are on the rise, and epidemiologicalstudies have shown that hyperlipidemia, hypertension and diabetes areclosely associated with atherosclerosis. More seriously, hyperlipidemia,hypertension and hyperglycemia often come together, which has graduallybecome one of the important health hazards worldwide.

As one type of commonly used CMM, raw Rehmannia glutinasa Libosch. isthe fresh tuber of Rehmannia glutinasa Libosch of Scrophulariaceaefamily with wide clinical applications, such as treating hematologicdiseases by enriching, cooling and regulating blood. As known in China,Rehmannia glutinasa Libosch. is usually used for treating such diseasesas heat diseases and deep-red tongue, interior heat and diabetes,efficiency of liver-yin and kidney-yin, hectic fever and so on. Inrecent years, many researches have been carried out on the chemicalcomposition and pharmacological action of Rehmannia glutinasa Libosch.It is reported that Rehmannia glutinasa Libosch. has various biologicalactivities on the systems of immune, endocrine and blood, thecardio-cerebral vascular system, the nervous system and so on. As themain components, phytosterols, carbohydrates, amino acids, iridoidglycosides such as catalpol and the like are included in Rehmanniaglutinasa Libosch.

After extensive studies and experiments, the present applicant foundthat as one type of main components in Rehmannia glutinasa Libosch.,iridoid glycosides such as catalpol and the like are highly polarcompounds with similar chemical structures, which have low chemical andthermal stability and are difficult to be extracted and separated. As aresult, there is no Rehmannia glutinasa Libosch. extract in the existingproducts that has a definite main effective composition, contains highcontent of catalpol, and has high stability and reliable efficacy inreducing blood glucose and blood lipid.

DISCLOSURE OF INVENTION

In medicine field, “effective fraction” of CMM refers to non-singlechemical components extracted from Chinese medicinal materials, naturalmateria medica and compounded Chinese materia medica, including totalflavonoids, total alkaloids, etc. One or more kinds of such chemicalcomponent should have a content of over 50% based on the total extractsand one or more kinds of such known chemical component should beregarded as effective components. And it is necessary for an effectivefraction as a whole to have clear chemical constitution (one kind orseveral kinds of substances) with its composition and content definiteand stable, and to have one to two kinds of definite main effectivecomponent.

An object of the present invention is to provide an effective fractionof Rehmannia glutinasa Libosch. that has reliable efficacy in reducingblood glucose, lipid levels and treating hematologic diseases such asleukaemia, and of which the effective component is specific and has highcontent and stability.

Another object of the present invention is to provide a method forpreparing the effective fraction of Rehmannia glutinasa Libosch., whichis advantageous over the prior art in respect of being reliable,repeatable, and simple for operation, and is suitable for producing onan industrial scale.

A further object of the present invention is to provide medicaments ofvarious dosage forms made from the effective fraction of Rehmanniaglutinasa Libosch.

Specifically, the present invention is implemented by the followingtechnical solutions:

The present invention provides an effective fraction of Rehmanniaglutinasa Libosch. that has reliable efficacy in reducing blood glucoseand lipid levels and treating hematologic diseases such as leukaemia,characterized in that the content of catalpol in the effective fractionis not lower than 50%, preferably 90-99.8%. The effective fraction ofRehmannia glutinasa Libosch. according to the present invention is whiteand non-odor crystal.

More preferably, the content of catalpol in the effective fraction ofRehmannia glutinasa Libosch. according to the present invention is 96%.

The effective fraction of Rehmannia glutinasa Libosch. according to thepresent invention is prepared by the following method:

Overground or underground parts of fresh Rehmannia glutinasa Libosch.were extracted with water or ethanol, and the resultant extract liquidwas concentrated to obtain extract; after the extract was dissolved withwater, the result solution was subjected to filtration; the filtrate wasloaded on a column packed with macroporous adsorption resin, followed byeluted with water until water eluate became clear, and then gradientlyeluted with ethanol; finally the eluate was recovered, concentrated anddried, thus the effective fraction of Rehmannia glutinasa Libosch.according to the present invention is obtained. The content of catalpolin such effective fraction should be not lower than 50%, and the optimumcontent was up to 90-99.8%.

In the present invention, the raw material used for extraction is theoverground stem and leaves or underground tuber of fresh Rehmanniaglutinasa Libosch. Extraction solvent is water or ethanol. Themacroporous adsorption resins are selected from non polar or weak polarresins, such as H103, D101, HP10, HP-20, HP-21, AB-8, D101 type, D201type, AB-8 type, HPD-100, HPD-200, HPD-100A and the like. The elution isgradiently eluted with water and ethanol (10-90%); and the eluate isconcentrated and dried under vacuum, with the temperature controlled at50-750° C.

A detailed method for preparing the effective fraction of Rehmanniaglutinasa Libosch. according to the present invention is shown below:

Sliced fresh Rehmannia glutinasa Libosch. is extracted at 60-80° C. with20-50% ethanol for 2-5 hours; then the ethanol is recovered and theextract liquid is concentrated to such an extent that there is no odorof ethanol to obtain extract; after the extract is dissolved with waterof 4-6 times the weight of the raw material, the resultant solution isfiltrated, and then the filtrate is loaded on a column packed withmacroporous adsorption resin of 1-3 times the weight of the rawmaterial; the column is firstly eluted with water until water eluatebecomes clear, followed by eluted with 10-40% ethanol of 30-50 times theweight of the raw material, then the finally the ethanol eluate isrecovered, concentrated and dried, thus the effective fraction ofRehmannia glutinasa Libosch. is obtained. The content of catalpol inthis effective fraction is 90-99.8%.

The most preferable method for preparing the effective fraction ofRehmannia glutinasa Libosch. according to the present invention is asfollows:

The fresh Rehmannia glutinasa Libosch. is sliced into sheets about 1 cmin thickness, and the sheets are extracted at 75° C. with 30% ethanolfor 3 hr; then the ethanol is recovered and the extract liquid isconcentrated to such an extent that there is no odor of ethanol toobtain extract; after the extract is dissolved with water of 5 times theweight of the raw material, the resultant solution is filtrated, andthen the filtrate is loaded on a column packed with H103 macroporousadsorption resin of twice the weight of the raw material; the column isfirstly eluted with water until water eluate becomes clear, followed byeluted with 20% ethanol of 40 times the weight of the raw material.Finally the ethanol eluate is recovered, concentrated, and dried at 60°C., thus the effective fraction of Rehmannia glutinasa Libosch. isobtained. The content of catalpol in this effective fraction is 96%.

The above-mentioned effective fraction of Rehmannia glutinasa Libosch.can be further made into clinically acceptable dosage forms bypharmaceutically conventional method, such as capsule, tablet, granule,pill, oral liquid preparation, dropping pill, suppository, injection andso on.

Study on Extraction Process

I. The Selection of Raw Material

Rehmannia glutinasa Libosch. is the tuber of Rehmannia glutinasa Liboschof Scrophulariaceae family, usually classified into two categories, i.e.raw Rehmannia glutinasa Libosch. and prepared Rehmannia glutinasaLibosch. These two categories differ in their functions and therapeuticapplications: raw Rehmannia glutinasa Libosch. has the efficacy ofclearing away heat and cooling the blood and nourishing Yin to promotethe production of body fluid due to its cold property; preparedRehmannia glutinasa Libosch. is used for nourishing Yin and enrichingblood and supplement essence and replenishing marrow. Catalpol is one ofthe main effective components in Rehmannia glutinasa Libosch., and haspharmaceutical effect such as reducing blood glucose and lipid levels,diuresis, moderating defecating and so on.

Our experiments showed that, when the harvested Rehmannia glutinasaLibosch. was dried by various methods such as freeze-drying,shade-drying, sun-drying or oven-drying, or was processed into preparedRehmannia glutinasa Libosch., the content of catalpol in them decreasedmore or less. Besides, the content of catalpol in fresh Rehmanniaglutinasa Libosch. decreased with the drying temperature increasing, andthe drying period prolonging, and the color of Rehmannia glutinasaLibosch. darkening. However, heating is not the sole factor affectingcatalpol content, and even under cryopreservation conditions, thecontent of catalpol in fresh Rehmannia glutinasa Libosch. alsodecreased.

Therefore, in the present invention, the raw material for extraction israw Rehmannia glutinasa Libosch., comprising its roots, stalks andleaves.

II. The Screen of Extraction Conditions

1. The Determination of Extraction Solvents

Solution I: Take two parts of 20 g of raw Rehmannia glutinasa Libosch.,one part was extracted with water of 10 times the weight of the rawmaterial for 1 h for 3 times, each time make up to the volume of 50 mL.Another part was extracted with 80% aqueous ethanol solution of 10 timesthe weight of the raw material for 1 h for 3 times, each time make up tothe volume of 50 mL. After a ten-fold dilution, these samples wereanalyzed by HPLC for measuring catalpol content.

Test Results: the extraction rate with water was 1.16%, and after thefirst extraction, 60% of catalpol was extracted; the extraction ratewith 80% ethanol was 2.08%, and after the first extraction, 78% ofcatalpol was extracted. So, alcohol extraction method was selected forextracting catalpol from raw Rehmannia glutinasa Libosch. because of itssuperior extraction rate.

Solution II: Take two parts of 20 g of raw Rehmannia glutinasa Libosch.,one part was extracted with water of 8 times the weight of the rawmaterial for 1 h for 3 times, each time make up to the volume of 50 mL.Another part was extracted with 30% aqueous ethanol solution of 8 timesthe weight of the raw material for 1 h for 3 times, each time make up tothe volume of 50 mL. After a ten-fold dilution, these samples wereanalyzed by HPLC for measuring catalpol content.

Test Results: the extraction rate of the dry extract with water was 30%,and the purity of catalpol was 95%; the extraction rate of the dryextract with 30% ethanol was 38%, and the purity of catalpol was 96%. Itcan be seen that extraction with both solvents could obtain catalpolwith high purity, but extraction with aqueous ethanol solution leaded tohigher extraction rate.

2. Concentration of the Ethanol for Extraction and Extraction Time

Table of factors level Condition A B C Level Temperature (° C.) Time(hr) Concentration (%) 1 65 3 30 2 70 2 50 3 75 1 70 Orthogonal Test andthe Results Extraction No. A B C D Rate (%) 1 1 1 1 1 0.38 2 1 2 2 20.33 3 1 3 3 3 0.36 4 2 1 2 3 0.40 5 2 2 3 1 0.32 6 2 3 1 2 0.39 7 3 1 32 0.40 8 3 2 1 3 0.36 9 3 1 2 1 0.37 K1 1.07 T = 3.31 K2 1.18 1.13 1.07K3 1.11 1.01 R 1.10 1.12 1.13 1.12 1.08 1.12 0.06 0.17 0.05 0.05

Conclusion: As is shown in the above table, the factor B had thegreatest effect on the test result, followed by the factor A and thenthe factor C, and among the factors B, A and C, B₁, A₃ and C₁ were themost preferable ones respectively. So the best extraction processcondition for Rehmannia glutinasa Libosch. was A₃B₁C₁, i.e. the bestextraction condition was that the temperature was 75° C., the extractiontime was 3 hr and the concentration of ethanol was 30%. However, othersolvents or ethanol solutions of other concentrations could also beapplicable for the extraction of the effective fraction of Rehmanniaglutinasa Libosch., but their performances were inferior.

3. The Selection of Resins

Non-polar Resins: H103, D101, HP10

Polar Resin: NKA-II

Weak-Polar Resin: AB8

To separate the main effective composition, i.e. catalpol from theextract liquid acquired above, different types of resins wereinvestigated: non polar resin H103, D101 and HP10, polar resin NKA-, andweak polar resin AB-8. Firstly, adsorption capacities and elution rateof these resins for catalpol were tested as follows: columns were packedwith these resins respectively; a certain amount of extract liquid wasloaded on the column to flow through the resin; eluates were collectedand analyzed for their catalpol content L₁ to value the adsorptioncapacities of different types of resins for catalpol. The smaller the L₁was, the higher adsorption capacity the resin had for catalpol in theextraction liquid, and vice versa. After adsorption, each resin that hadhigh adsorption capacity was eluted in the same eluting method, and theeluates were collected and analyzed for their catalpol content L₂. Thehigher the L₂ was, the better the elution effect was. The detailedresults was shown in the table below:

Results of Resin Performance Resin Type H103 D101 NKA-II AB-8 HP10HPD-200 L₁ (%) 2.1 3.3 93 2.0 3.5 2.8 L₂ (%) 94 95.5 — 97 96.8 98.1

As shown in the above table, resin NKA-II failed to separate catalpolfrom the extract liquid of Rehmannia glutinasa Libosch., but resinsH103, D101, AB-8, HP10 and HPD-200 were able to separate catalpol withcontent up to 90% in the final products. Our further investigationshowed that other resins, such as HP-20, HP-21, AB-8, D101, D201,HPD-100 and HPD-100A were also suitable for separating catalpol.

4. Concentration of Ethanol for Elution

After the extract liquid was filtrated through macroporous resin H103,besides catalpol, saccharides and other impurities in Rehmanniaglutinasa Libosch. were also adsorbed on the resin, and moreover therewere other free impurities. So, water was firstly used to elute thesaccharides and the free impurities until eluate became clear. Afteranalyzed by HPLC, it was found that the water eluate did not containcatalpol, which suggested that catalpol was still left on the resin.Then the adsorbed catalpol was gradiently eluted with aqueous ethanolsolutions with different concentrations of 10%, 20%, 30%, 40%, 50% and60%, and the flow rate was 0.5-5 ml/cm²·min. After analysis, no catalpolwas found in the eluate obtained by eluting with aqueous ethanolsolutions having a concentration of above 40%, which indicated thatcatalpol was liable to decompose in ethanol. And the eluate obtained byeluting with aqueous ethanol solution having a concentration of 20% wasfound to contain the highest yield and purity of catalpol. Therefore,aqueous ethanol solution having the concentration of 20% was used as theeluate, and its amount should be 40 times as much as the raw material.To obtain the final product, the eluate needed to be furtherconcentrated and dried under vacuum, and the optimization method wasshown below.

5. The Selection of Drying Method and Temperature

In order to investigate the thermostability of the main effectivecomposition, i.e. catalpol, in the effective fraction of Rehmanniaglutinasa Libosch., high temperature test was performed on dried samplesof the effective fraction under the conditions listed in the followingtable, which are used adopted in the high temperature test onmedicaments in China.

High Temperature Test Storage Temperature 60□ Storage Period 0 5 days 10days Catalpol Content 96% 94% 88%

Then the dried samples of the effective fraction were extracted with 30%aqueous ethanol solution for 5 h, and it was found that catalpol contentin the extract did not decrease with the elapse of time but increasedgradually. Lastly, in the process of concentration and drying, drying at95° C. and drying at 60° C. under vacuum had greater effects on thecatalpol content: the catalpol content decreased to 8-12% of theoriginal content in the former and to 90-98% of the original content inthe latter. In conclusion, catalpol was stable in the process of solventextraction, but it should be noted that, the temperature must not be toohigh in concentration and drying process.

Identification and Content Determination of the Effective Fraction ofRehmannia glutinasa Libosch. According to the Present Invention

1. Property

The effective fraction of Rehmannia glutinasa Libosch. according to thepresent invention is white to pale yellow crystal powder with fragrantodor and slightly sweet taste. It readily dissolves in water andmethanol, slightly dissolves in ethanol and acetone, and does notdissolve in ether. Its melting point is 207-209° C., and it decomposeswhile melting.

2. Identification

1) Chromatograms recorded under the item of the content determinationshowed that the retention time of the main peak of the sample wasaccordance with that of the standard sample.

2.) The infrared absorption spectra of the sample were consistent withthe standard spectra.

3. Content Determination

The determination was made according to the HPLC method as described inAppendix VI.D in the Pharmacopoeia of People's Republic of China of 2005version.

Chromatographic conditions and system suitability test: packingmaterial: octadecylsilyl silica gels; mobile phase: acetonitrile-0.1%aqueous phosphoric acid (1:99); detection wavelength: 210 nm. Thetheoretical plate number calculated with catalpol must not be lower than3000.

Preparation of Standard solution: accurately weighted standard catalpolwas dissolved in methanol to form a solution having a concentration of0.3 mg/ml.

Preparation of Sample solution to be measured: an accurately weightedsample in the range of about 0.3 g was added to a 100 ml volumetricflask. The sample dissolved with methanol was diluted until reaching thecalibration mark, shaken up and filtrated through micropore membrane(0.45 μm) to obtain the sample solution to be measured.

Test method: 10 μl of the standard solution and the sample solution tobe measured were taken separately, followed by being injected intoliquid chromatography and being measured.

Conclusions: the content of catalpol in the effective fraction ofRehmannia glutinasa Libosch. according to the present invention was inthe range of 90-99.8% with an average of 96%.

Pharmacology Experiments

In order to prove the advantage and progress of the effective fractionof Rehmannia glutinasa Libosch. according to the present invention overthe existing similar products, comparisons including pharmacologyexperiments were made between the effective fraction of Rehmanniaglutinasa Libosch. according to the present invention and the similarRehmannia glutinasa Libosch. extracts prepared according to the priorart by the present application.

1. Experimental Materials

1.1 Samples

Sample to be tested: the effective fraction of Rehmannia glutinasaLibosch. according to the present invention, labeled as A;

Positive Control:

The total glycoside extract from the stalks and leaves of Rehmanniaglutinasa Libosch. prepared according to the disclosure ofCN200410062245, labeled as B,

Rehmannia glutinasa Libosch. extract obtained according to the methoddisclosed in Technologies for Purification and Separation of IridoidGlycosides from Dried Rehmannia Root, Herald of Medicine, October 2003,22(10), labeled as C,

Rehmannia glutinasa Libosch. extract obtained according to the methoddisclosed in Study on Separation and Purification of Catalpol inRehmannia glutinasa Libosch., China Journal of Chinese Materia Medica,June 2004, 29(6), labeled as D,

Rehmannia glutinasa Libosch. extract (dark brown powder of iridoidglycosides in raw Rehmannia glutinasa Libosch.) obtained according tothe method disclosed in Separation and Purification of Carbohydrates andGlycosedes in Rehmannia glutinasa Libosch. Using Macroposous Resin,Journal of Chinese Medicinal Materials, March 2003, 26(3), labeled as E,

Rehmannia glutinasa Libosch. extract obtained according to the methoddisclosed in Technological Process for Catalpol of Rehmannia glutinasaLibosch, Lishizhen Medicine and Materia Medica Research, 2000, 11(4),labeled as F;

dimethyl diguanidine (hereinafter abbreviated as DD): produced byZhengan (Tianjin) medicine Company Ltd.

alloxan: produced by Sigma, USA

kits for detecting glucose: produced by Beijing Xinde Institute ofBiological Products

1.2 Main Instruments

CHEM 300 semi-automatic biochemical analyzer (Germany)

GT-1640 Jingdu Blood Glucose Meter (Arkray)

1.3 Animals

Male Kunming mice weighing 25-29 g

Raising condition: The mice were housed in a laboratory animal room of2nd grade that was ventilated regularly with the temperature controlledat 20-24° C. Before the test began, their food taking, activities andfeces were observed for one week, and normal mice were selected forfurther test.

2. Experimental Method and Result

2.1 Preparation of Model Mice Suffered from Alloxan-InducedHyperglycemia

Male mice were selected and firstly fasted for 6 hr, and alloxan wasinjected into tail vein at a dose of 60 mg/kg. After 72 hr, the micehaving blood glucose values >11.1 mol/L were selected as experimentalanimals and grouped.

2.2 Experimental Method:

The alloxan-induced hyperglycemic mice were randomly divided into groupslabeled as normal control, model control, low-dose A, mid-dose A,high-dose A, DD, B, C, D, E and F. Subsequently, intragastricadministration was performed for 15 consecutive days. 16 hours after thelast administration, the mice were fasted for 8 hr. Blood glucose valuesbefore administration, and at 1 hour after administration for 5 days, 10day and 15 days were tested respectively on blood collected from theinguinal vein, and the results were as follows:

TABLE 1 Effect of the Effective Fraction/Extracts of Rehmannia glutinasaLibosch. on Alloxan-Induced Hyperglycemic Mice Blood Glucose Value(mmol/L) Dose Mice 0 5 10 15 (mg/Kg) Number day days days days Normal 225.5 6.4 6.4 5.8 Control Model 22 19.6^(ΔΔ) 24.3^(ΔΔ) 24.9^(ΔΔ) 21.3^(ΔΔ)Control Low-Dose A 125 22 19.5 18.9 17.5 15.3 Mid-Dose A 250 22 19.715.2 12.8** 9.2* High-Dose A 500 22 20.0 14.2** 11.9** 8.7** B 250 2220.1 18.7 16.4 15.9 C 250 22 19.8 19.1 15.7 14.8 D 250 22 19.6 18.2 17.215.6 E 250 22 19.8 18.6 16.1 14.9 F 250 22 20.5 19.9 16.3 14.6 DD 500 2219.7 12.5** 13.2** 14.0** **P < 0.01, *P < 0.05 (compared with the modelcontrol); ^(ΔΔ)P < 0.01 (compared with the normal control)

2.3 Preparation of Model Mice with Type II Diabetes

Forty male and female KK diabetes mice weighing about 40 g wereseparately housed at a temperature of 20-40° C. and fed on a fat-heavydiet and arbitrary amount of water. After being fast food but not fastwater for 2 hr, 10 μl blood collected from the tail veins was tested forblood glucose values, and mice having blood glucose value ≧8.0 mmol/Lwere selected.

2.4 Experimental Method

The mice obtained from the above step were randomly divided into groupslabeled as model control, low-dose A, mid-dose A, high-dose A, DD, B, C,D, E and F. Subsequently, intragastric administration was performed oncea day for 15 consecutive days. Blood glucose values beforeadministration, and at one hour after administration for 5 days, 10 dayand 15 days (fast food but not fast water for two hours after lastadministration) were tested respectively, and the results were asfollows:

TABLE 2 Effect of the Effective Fraction/Extracts of Rehmannia glutinasaLibosch. on the Blood Glucose in the Spontaneous Hyperglycemic KK MiceDose Mice Blood Glucose Valure (mmol/L) Sample (mg/kg) Number 0 day 5days 10 days 15 days Model Control 0.2 ml/kg 11 13.40 ± 2.53 14.58 ±2.95 15.92 ± 3.90 15.35 ± 4.66 Low-Dose A 125 11 12.71 ± 2.25 10.97 ±0.66** 11.24 ± 1.99*  9.77 ± 3.12*** Mid-Dose A 250 12 13.11 ± 4.81 9.96 ± 1.78**  8.12 ± 3.75**  6.33 ± 2.39** High-Dose A 500 12 13.05 ±5.01  9.60 ± 2.29**  7.25 ± 1.35***  5.12 ± 1.93*** B 250 12 13.89 ±3.11 11.01 ± 2.19 10.13 ± 1.95  9.15 ± 3.18 C 250 12 12.89 ± 2.99 10.96± 4.097 10.54 ± 2.10  8.97 ± 5.03 D 250 12 14.11 ± 3.22 12.13 ± 2.7310.35 ± 1.97  9.11 ± 3.23 E 250 12 13.54 ± 3.65 11.75 ± 2.29  9.43 ±2.10  9.01 ± 3.72 F 250 12 14.13 ± 2.17 12.13 ± 4.21 10.09 ± 3.41  8.99± 3.57 DD 500 11 13.36 ± 2.85 11.16 ± 3.19*** 10.03 ± 2.11***  8.84 ±4.51*** Note: compared with the model control: *p < 0.05, **p < 0.01,***p < 0.001

3. Experimental Results

3.1 As shown in Table 1, the model control group significantly differedfrom the normal control group in respect of the blood glucose values,which indicated the model was successfully established. Compared withthe model control group, the positive medicine dimethyl diguanidinegroup and the mid-dose A group showed dramatic decrease in the fastingblood glucose value after 5 days administration (P<0.01), and thehigh-dose A group and the mid-dose A group also showed dramatic decreasein the fasting blood glucose value after 10 days administration(P<0.01). The blood glucose values of the dimethyl diguanidine groupfirst decreased and then increased during the whole administrationperiod, and those in the mid-dose A group and the high-dose A groupdecreased steadily. So conclusions can be made that, mid-dose A groupand high-dose A group had good hypoglycemic effect, wherein a gooddose-effect relationship can be observed, and their hypoglycemic effectwas more obvious in comparison with the samples B, C, D, E and F fromthe experimental process and experimental data.

3.2 As shown in Table 2, the KK mice model control group wassignificantly lower than the groups of dimethyl diguanidine, low-dose A,mid-dose and high-dose in respect of the fasting blood glucose valueafter 5 days administration (P<0.01); the groups of mid-dose andhigh-dose were significantly lower than the groups of the model control,B, C, D, E and F in respect of the fasting blood glucose value after 10days administration (P<0.01) and after 15 days administration (P<0.01).So conclusions can be made that, mid-dose A group and high-dose A grouphad significant hypoglycemic effect in contrast with the other controlmedicines.

3.3 It can be found in the above test that the Rehmannia glutinasaLibosch. extract according to the present invention was also able toreduce blood lipid, and its lipid-decreasing effect was obvious ascompared with other control medicines.

Experimental Study on Catalpol and the Rehmannia glutinasa Libosch.Extract for their Effect of Inducing Apoptosis in Acute NonlymphocyticLeukemia Cell

In order to investigate catalpol and the Rehmannia glutinasa Libosch.extract according to the present invention for their effect of inducingapoptosis in leukemia cells as well as the mechanism, bone marrows andHL260 cells from 15 patients with acute nonlymphocytic leukemia (ANLL)were mixed with catalpol and co-cultivated for 24 hr, and then differenttest methods including cytomorphologic means, DNA gel electrophoresisand DNA fragments percentage analysis were adopted to observe the effecton apoptosis in leukemia cells. Then 8 samples of bone marrow cellsbefore and after above treatment were detected for the proteinexpression of genes bcl22 and c2myc (gene families of B-celllymphoma/leukemia 22 and cell 2 oncogene) by immunohistochemical method.The result showed that, typical apoptotic morphology andtrapezoid-shaped DNA band were observed in each treated sample, butthere was no such phenomenon in the control sample; the proteinexpression of genes bcl22 and c2myc of the bone marrow cells treatedwith catalpol and the Rehmannia glutinasa Libosch. extract was obviouslylowered.

Conclusion: catalpol and the Rehmannia glutinasa Libosch. extract wereable to induce apoptosis in ANLL cell and HL260 cell, and one possiblemechanism of the apotosis induction was that catalpol inhibited orlowered the protein expression of genes bcl22 and c2myc.

DNA Fragments Percentage at 24 Hrs after Treatment with Catalpol:

Group n control HHT(10 − 7 mol · L⁻¹ Ara2c (10 μg · ml⁻¹) PL (20 μg ·ml⁻¹) HA ANLL cell 24.2 ± 4.3 38.1 ± 5.1 40.1 ± 8.2 39.5 ± 7.9 46.2 ±6.4 15 HL260 cell 10.2 ± 0.2 26.1 ± 1.2 28.2 ± 1.4 30.3 ± 1.2 34.3 ± 1.64DNA Fragments Percentage after Treating with the Extract for 24 Hrs

Group n control HHT(10 − 7 mol · L⁻¹) Ara2c (10 μg · ml⁻¹) PL (20 μg ·ml⁻¹) HA ANLL cell 22.1 ± 3.8 36.2 ± 5.0 39.7 ± 7.9 37.3 ± 7.9 45.2 ±5.8 15 HL260 cell 10.0 ± 0.1 25.3 ± 1.0 27.0 ± 1.1 30.1 ± 1.2 33.1 ± 1.44 Note: P < 0.05 for each treatment group in comparison with the controlgroup; P > 0.05 among all the sample groups

The above result indicated that the Rehmannia glutinasa Libosch. extractaccording to the present invention was effective for acutenonlymphocytic leukemia as well as leukemia of other types, and thus canbe used to prepare medicaments for treating leukemia.

Safety Investigation on the Effective Fraction of Radix Rehmannia

After toxicological experiment on animals, it was found that the maximumamount of 35.2 g/kg by oral administration did not lead to death of theanimal in acute toxicity test, and the LD50 value of intraperitonealinjection administration was 12.15-16.46 g/kg.

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples are preferable embodiments for carrying out thepresent invention, but they are not intended to limit the scope of thepresent invention.

Example 1 The Effective Fraction of Rehmannia glutinasa Libosch.According to the Present Invention

The fresh Rehmannia glutinasa Libosch. was sliced into sheets about 1 cmin thickness, and the sheets were extracted at 72° C. with 28% ethanolfor 3 hours; then the ethanol was recovered and the extract liquid wasconcentrated to such an extent that there was no odor of ethanol toobtain the extract; after the extract was dissolved with water of 5times the weight of the raw material, the resultant solution wasfiltrated, and then the filtrate was loaded on a column packed with H103macroporous adsorption resin of twice the weight of the raw material;the column was firstly eluted with water until the water eluate becameclear, followed by eluted with 20% ethanol of 40 times the weight of theraw material; finally the ethanol eluate was recovered, concentrated,and dried at 55° C. to obtain the effective fraction of Rehmanniaglutinasa Libosch. The content of catalpol in this effective fractionwas 94.8%.

Example 2 The Effective Fraction of Rehmannia glutinasa Libosch.According to the Present Invention

The fresh Rehmannia glutinasa Libosch. was sliced into sheets about 1 cmin thickness, and the sheets were extracted at 75° C. with 30% ethanolfor 3 hours; then the ethanol was recovered and the extract liquid wasconcentrated to such an extent that there was no odor of ethanol toobtain the extract; after the extract was dissolved with water of 5times the weight of the raw material, the resultant solution wasfiltrated, and then the filtrate was loaded on a column packed with H103macroporous adsorption resin of twice the weight of the raw material;the column was firstly eluted with water until the water eluate becameclear, followed by eluted with 20% ethanol of 40 times the weight of theraw material; finally the ethanol eluate was recovered, concentrated,and dried at 60° C. to obtain the effective fraction of Rehmanniaglutinasa Libosch. The content of catalpol in this effective fractionwas 96%.

Example 3 The Effective Fraction of Rehmannia glutinasa Libosch.According to the Present Invention

The fresh Rehmannia glutinasa Libosch. was sliced into sheets about 1 cmin thickness, and the sheets were extracted at 80° C. with 45% ethanolfor 2.5 hours; then the ethanol was recovered and the extract liquid wasconcentrated to such an extent that there was no odor of ethanol toobtain the extract; after the extract was dissolved with water of 7times the weight of the raw material, the resultant solution wasfiltrated, and then the filtrate was loaded on a column packed with H103macroporous adsorption resin of three times the weight of the rawmaterial; the column was firstly eluted with water until the watereluate became clear, followed by eluted with 20% ethanol of 40 times theweight of the raw material; finally the ethanol eluate was recovered,concentrated, and dried at 75° C. to obtain the effective fraction ofRehmannia glutinasa Libosch. The content of catalpol in this effectivefraction was 93.8%.

Example 4 Capsules of the Effective Fraction of Rehmannia glutinasaLibosch. According to the Present Invention

Capsules can be prepared by directly filling the effective fraction ofRehmannia glutinasa Libosch. according to the present invention intocapsule shells, or by mixing it with one or more pharmaceuticalexcipients for capsules and then filling them into capsule shells.

Example 5 Tables of the Effective Fraction of Rehmannia glutinasaLibosch. According to the Present Invention

Tables can be prepared by using the effective fraction of Rehmanniaglutinasa Libosch. according to the present invention together with oneor more excipients for tablets by conventional methods for preparingtablets. The usable excipients include but are not limited to starch,dextrin, lactose, sugar powder, calcium sulfate, microcrystallinecellulose, mannitol, magnesium stearate, gelatin, arabic gum, methylcellulose, sodium carboxymethyl cellulose, low-substituted hydroxypropylcellulose, hydroxypropyl methylcellulose, povidone, sodium alginate,polyethylene glycol, cross-linked sodium carboxymethyl cellulose, sodiumcarboxymethyl starch, talc and micro silica.

Example 6 Granules of the Effective Fraction of Rehmannia glutinasaLibosch. According to the Present Invention

Granules can prepared by using the effective fraction of Rehmanniaglutinasa Libosch. according to the present invention and one or moreexcipients for granules by conventional methods for preparing granules.The usable excipients include but are not limited to sucrose, dextrin,citric acid and sodium citrate.

The invention claimed is:
 1. An effective fraction of Rehmanniaglutinasa Libosch. for reducing blood glucose and lipid levels, whereinthe effective fraction of Rehmannia glutinasa Libosch. containsnon-single chemical components produced from Rehmannia glutinasaLibosch., wherein the non-single chemical components produced includecatalpol in an amount of 50% or more, and wherein the non-singlechemical components of the effective fraction are produced by extractingfresh Rehmannia glutinasa Libosch. with water or ethanol, andconcentrated to obtain an extract, dissolving the extract with water toobtain a solution, loading the solution on a column packed withmacroporous adsorption resin, eluting the loaded solution with ethanol;and recovering the ethanol eluate, wherein the effective fraction ofRehmannia glutinasa Libosch. significantly reduces blood glucose levelsin comparison to controls.
 2. An effective fraction of Rehmanniaglutinasa Libosch. for reducing blood glucose and lipid levels inaccordance with claim 1, characterized in that the content of catalpolin the effective fraction of Rehmannia glutinasa Libosch. is in a rangeof 90%-99.8%.
 3. The effective fraction of Rehmannia glutinasa Libosch.for reducing blood glucose and lipid levels according to claim 2,characterized in that the content of catalpol in the effective fractionof Rehmannia glutinasa Libosch. is 96%.
 4. A preparation of Rehmanniaglutinasa Libosch. for reducing blood glucose and lipid levels inaccordance with claim 1, characterized in that the preparation includespharmaceutical excipients added to the effective fraction of Rehmanniaglutinasa Libosch.
 5. The preparation of Rehmannia glutinasa Libosch.according to claim 4, characterized in that the preparation is in theform of tablet, capsule, granule or pill.
 6. The effective fraction ofRehmannia glutinasa Libosch. for reducing blood glucose and lipid levelsaccording to claim 1, wherein said macroporous adsorption resin is H103,D101, HP10, HP-20, HP-21, AB-8, D101, D201, HPD-100, HPD-200 orHPD-100A; the elution is gradiently eluted with 10-90% ethanol, andwherein the ethanol eluate is concentrated and dried under vacuum at atemperature in the range 50-70° C.
 7. The effective fraction ofRehmannia glutinasa Libosch. for reducing blood glucose and lipidlevels, according to claim 1, wherein the content of catalpol in theeffective fraction of Rehmannia glutinasa Libosch. is in a range of90%-99.8%, and fresh Rehmannia glutinasa Libosch. is extracted at 60-80°C. with 20-50% ethanol for 2-5 hours; the extract is dissolved withwater of 4-6 times the weight of the fresh Rehmannia glutinasa Libosch.,the macroporous adsorption resin is 1-3 times the weight of the freshRehmannia glutinasa Libosch.; 10%-40% ethanol of 30-50 times the weightof fresh Rehmannia glutinasa Libosch. is used to elute the solution, andwherein the recovered ethanol eluate is concentrated and dried at 50-70°C.
 8. The effective fraction of Rehmannia glutinasa Libosch. forreducing blood glucose and lipid levels according to claim 7, whereinthe content of catalpol in the effective fraction of Rehmannia glutinasaLibosch. is 96%.
 9. The effective fraction of Rehmannia glutinasaLibosch. for reducing blood glucose and lipid levels according to claim7, wherein the fresh Rehmannia glutinasa Libosch. is extracted at 75° C.with 30% ethanol for 3 hours; the extract is dissolved with water of 5times the weight of the fresh Rehmannia glutinasa Libosch., themacroporous adsorption resin is twice the weight of the fresh Rehmanniaglutinasa Libosch.; 20% ethanol of 40 times the weight of freshRehmannia glutinasa Libosch. is used to elute the solution, and whereinthe recovered ethanol eluate is concentrated and dried at 60° C.
 10. Theeffective fraction of Rehmannia glutinasa Libosch. of claim 1, whereinthe effective fraction is produced from roots, stalks, and leaves ofRehmannia glutinasa Libosch.
 11. The effective fraction of Rehmanniaglutinasa Libosch. of claim 10, wherein the effective fraction is awhite to pale-yellow powder.
 12. The effective fraction of Rehmanniaglutinasa Libosch. of claim 1, wherein the effective fraction inducesapoptosis in leukemia cells.
 13. The effective fraction of Rehmanniaglutinasa Libosch. of claim 1, wherein the effective fractionsignificantly reduces blood glucose levels at a dose equivalent to atleast 250 mg/kg of the effective fraction in mice.
 14. The effectivefraction of Rehmannia glutinasa Libosch. of claim 13, wherein theeffective fraction is in a dosage form suitable for administering a doseequivalent to at least 250 mg/kg of the effective fraction in mice. 15.The effective fraction of Rehmannia glutinasa Libosch, of claim 13,wherein the effective fraction significantly reduces blood glucoselevels at a dose equivalent to at least 500 mg/kg of the effectivefraction in mice.
 16. The effective fraction of Rehmannia glutinasaLibosch. of claim 15, wherein the effective fraction is in a dosage formsuitable for administering a dose equivalent to at least 500 mg/kg ofthe effective fraction in mice.
 17. An effective fraction of Rehmanniaglutinasa Libosch. for reducing blood glucose and lipid levels, whereinthe effective fraction of Rehmannia glutinasa Libosch. containsnon-single chemical components produced from Rehmannia glutinasaLibosch., wherein the non-single chemical components produced includecatalpol in an amount of 50% or more, and wherein the non-singlechemical components of the effective fraction are produced by extractingthe roots, stalks, and leaves of fresh Rehmannia glutinasa Libosch. withwater or ethanol, and concentrated to obtain an extract, dissolving theextract with water to obtain a solution, loading the solution on acolumn packed with macroporous adsorption resin, eluting the loadedsolution with ethanol; and recovering the ethanol eluate, wherein theeffective fraction of Rehmannia glutinasa Libosch. is a white topale-yellow powder having a melting point of about 207-209° C. andwherein the effective fraction of Rehmannia glutinasa Libosch.significantly reduces blood glucose levels in comparison to controls.